RUNX3是RUNX轉錄因子家族的其中一位成員。RUNX轉錄因子家族是負責調控細胞的增殖和分化。最近研究表明RUNX3可能在造血過程中扮演其中一個角色。可是，它在髓系細胞中的調節角色依然未明。此前，我們發現在核心結合因子急性骨髓性白血病中的融合蛋白RUNX1-ETO和CBFB-MYH11會抑制RUNX3基因表達，並且RUNX3表達水平對兒童急性骨髓性白血病的預後有顯著影響。本研究的目的是要調查RUNX3在成人急性骨髓性白血病的預後價值，並透過闡明RUNX3的轉錄調節去了解其在髓系細胞分化扮演的角色。 / 首先，我們透過實時定量聚合鏈反應去量化在174個成人急性骨髓性白血病的患者骨髓中的RUNX3表達，從而調查RUNX3表達與成人急性骨髓性白血病預後的關係。我們發現低RUNX3表達與較好預後的核型(P=0.045)，NPM1基因突變(P=0.014) 和較年青患者(P=0.084) 有關聯。在存活分析中，我們把有完整生存數據的非急性前骨髓性白血病病人分成高RUNX3表達和低RUNX3表達兩組。在成人急性骨髓性白血病中，高RUNX3表達和較差整體存活率(OS) (P=0.011)和無事件存活率(EFS) (P=0.003)有顯著的關聯，這和我們在兒童急性骨髓性白血病所觀察的一致。高RUNX3表達和較差存活率的關係在有野生型FLT3基因的病人中更為明顯(OS, P=0.004; EFS, P=0.001)。由於低RUNX3表達和較好預後核型有關聯，我們進一步只對擁有較差預後核型的病人作將存活分析，發現RUNX3表達仍是影響EFS的一個顯著因素(P=0.017)。在多元分析中，高RUNX3表達在所有病人(EFS, P=0.026, HR=2.433, 95%CI = 1.114-5.356)，野生v 型FLT3基因的病人(OS, P=0.016, HR=4.830, 95%CI = 1.335-17.481; EFS, P=0.007, HR=4.103, 95%CI = 1.480-11.372)和較差預後核型的病人(EFS, P=0.024,HR=2.339, 95%CI = 1.117-4.896) 中都是一個獨立的不利預後因素。 / 接著，我們研究RUNX3基因的表達調控。我們鑒定出一個最小啟動子區對於在髓系細胞的基因表達有關鍵作用。透過預測啟動子區和轉錄因子結合位點的分析，顯示這個活性區域含有PU.1，AP-1和Sp1轉錄因子結合位點。我們透過報告基因系統研究，染色質免疫沈澱技術及電泳遷移率改變分析去闡明PU.1，c-Jun及Sp1和相對的轉錄因子結合位點參與RUNX3基因的表達調控。我們進一步透過PU.1基因剔除去證實RUNX3是PU.1的直接下遊靶基因並發現PU.1與RUNX3表達在急性骨髓性白血病人中呈正相關性。 / 由於RUNX3基因表達受到PU.1, c-Jun及Sp1的控制，我們繼續研究RUNX3在髓系細胞分化的功用。我們透過實時定量聚合鏈反應及流式細胞儀檢測發現RUNX3過度表達誘導K562細胞株作單核細胞及粒細胞分化。RUNX3能激活髓系基因的啟動子。它在成熟髓系細胞的表達水平明顯比血幹細胞為高。根據以上結果，RUNX3也許在單核細胞及粒細胞分化中有一定功能。但是，有別於其他癌細胞，RUNNX3不能在髓系細胞誘導細胞凋亡和周期阻滯。 / 總括而言，RUNX3表達在成人急性骨髓性白血病中是一個獨立的預後因素。除此之外，本研究表明RUNX3受到PU.1，c-Jun及Sp1的表達調控並在單核細胞及粒細胞分化中有一定功能。 / RUNX3 is a member of Runt-related domain (RUNX) transcription factor family, which regulates cell proliferation and differentiation. Recent studies have suggested a role of RUNX3 in hematopoiesis. However, its regulatory function in myeloid cells remains unclear. Our group previously showed that RUNX3 expression was repressed by the fusion proteins RUNX1-ETO and CBFB-MYH11 in core-binding factor acute myeloid leukemia (CBF-AML) and had prognostic implication in childhood AML patients. The aim of this study is to investigate the prognostic value of RUNX3 in adult AML patients and its role in myeloid differentiation by elucidating its transcriptional control. / To investigate the relationship between RUNX3 expression and prognosis of adult AML, RUNX3 expression in the diagnostic bone marrow samples from 174 adult AML patients were quantified by real time quantitative PCR (RQ-PCR). Low RUNX3 expression was found to be associated with favorable cytogenetic group (P=0.045), NPM1 mutations (P=0.014) and younger age (P=0.084). For the survival analysis, 110 non-acute promyelocytic leukemia (non-APL) patients with complete survival data were dichotomized into high and low expression groups. Concordant with our previous observation in childhood AML, a significant association between high RUNX3 expression and poorer overall survival (OS) (P=0.011) and event-free survival (EFS) (P=0.003) was observed. The association between high RUNX3 expression and poorer survival was further strengthened in patients with wild-type FLT3 (P=0.004 and 0.001 for OS and EFS respectively). Since low RUNX3 expression was associated with favorable cytogenetics, the analysis was next restricted to patients with non-favorable cytogenetics and RUNX3 expression remained as a significant factor for EFS (P=0.017). In multivariate analysis, high RUNX3 expression was an independent adverse prognostic factor in the whole cohort (EFS, P=0.026, HR=2.433, 95%CI = 1.114-5.356), patients with wild-type FLT3 (OS, P=0.016, HR=4.830, 95%CI = 1.335-17.481; EFS, P=0.007, HR=4.103, 95%CI = 1.480-11.372) and patients with non-favorable genetics (EFS, P=0.024,HR=2.339, 95%CI = 1.117-4.896). / Next, the transcriptional regulation of RUNX3 in myeloid cells was investigated. A minimal promoter region was identified to be critical for myeloid-specific promoter activity. Sequence analysis of the fragment revealed potential transcription factor binding sites for PU.1, AP-1 and Sp1.The involvement of these putative binding sites and corresponding transcription factors in transcriptional regulation of RUNX3 was demonstrated by promoter reporter assay, chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA).Furthermore, PU.1 knockdown in U937 cells confirmed RUNX3 was a direct downstream target of PU.1 and a positive correlation between PU.1 and RUNX3 expression was observed in AML patient samples. / As RUNX3 was shown to be transcriptionally regulated by PU.1, c-Jun and Sp1, a role of RUNX3 in myeloid differentiation was postulated. Overexpression of RUNX3 induced both monocytic and granulocytic markers in K562 myeloid cells as detected by flow cytometry and RQ-PCR. RUNX3 was also found to activate myeloid-specific gene promoters and its expression was significantly higher in mature myeloid cells than in hematopoietic stem cells. This suggested a role of RUNX3 in both monocytic and granulocytic differentiation. However, unlike in other solid tumors, RUNX3 did not induce apoptosis and cell cycle arrest in myeloid cells. / In conclusion, RUNX3 expression was an independent prognostic factor in adult AML. Furthermore, our findings showed that RUNX3 was transcriptionally regulated by the master myeloid regulator PU.1 along with c-Jun and Sp1 and implicated a role in monocytic and granulocytic differentiation. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Kwan, Tsz Ki. / Thesis (Ph.D.) Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 171-202). / Abstracts also in Chinese.

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Pathology / Master / Master of Philosophy

published_or_final_version / Medicine / Master / Master of Philosophy

Doctorat en Sciences / info:eu-repo/semantics/nonPublished

TAL-1, ectopically expressed in 60% of T-cell acute lymphoblastic leukemia (T-ALL) patients, may contribute to poor chemotherapy response. This research sought to determine if TAL-1 influences expression of proteins involved in the NF-kB signaling pathway and thus, resistance to cell death. NF-kB, IKKy, and TRAF-2 expression levels were found to be TAL-1 dependent. Cell death levels were higher in staurosporine-treated cells compared to tumor necrosis factor a-treated or dual-treated cells. TAL-1, NF-kB, IKKy, and TRAF-2 expression levels were elevated in tumor necrosis factor a-treated cells and reduced in staurosporine-treated or dual treated cells compared to untreated cells. These results suggest TAL-1 influences expression of proteins involved in the NF-kB signaling pathway, thus inducing an anti-apoptotic response in the cell. / Department of Biology

Cancer has been a disease, which has generated intense research interest for many years. Misexpression of two oncoproteins, TAL 1 and LMO 1, has been found to help induce a particular type of leukemia, called T-cell acute lymphoblastic leukemia (T-ALL). Presently, it is not completely understood how these proteins induce leukemogenesis or what other cellular proteins they interact with to drive this progression. In this study, a series of experiments were conducted to identify downstream targets of TALI and LMO1. Using retroviral gene transfer, both genes were introduced, either singly or in combination, into a murine T-cell line called AKR-DP-603. Empty vectors were introduced as controls. In order to assay the effects of TALI and LMO I expression on expression of other proteins, a series of Western blots were completed on all populations of engineered cells. It was determined that there were differences in expression of Bcl-2 and p16 as indicated by differences in band intensities on the blots. This is important because it implies an effect on protein levels by TAL 1 and LMO 1. However, there were no differences in protein expression levels for Bax or cyclin D1. This suggests that TAL1 and LMOI do not have any regulatory effects on these proteins. In addition, apoptotic assays were completed on all populations of cells. The results of both a TUNEL assay and ethidium bromide/acridine orange staining protocol showed TAL1- and LMO1expressing cells to have an increase in cell survival under starvation conditions and a lower frequency of apoptosis. Statistical analysis verified significant difference in the apoptosis assays. The data suggests an up-regulation of anti-apoptotic proteins. The finding of this research allow a clearer understanding of the process of leukemogenesis and may lead to a development of better cancer treatments. / Department of Biology

Indiana University-Purdue University Indianapolis (IUPUI) / Juvenile Myelomonocytic Leukemia (JMML) is rare, fatal myeloproliferative disease (MPD) affecting young children, and is characterized by expansion of monocyte lineage cells and hypersensitivity to Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) stimulation. JMML is frequently associated with gain-of-function mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase, Shp2. Activating Shp2 mutations are known to promote hyperactivation of the Ras-Erk signaling pathway, but Akt is also observed to have enhanced phosphorylation, suggesting a potential role for Phosphatidylinositol-3-Kinase (PI3K)-Akt signaling in mutant Shp2-induced GM-CSF hypersensitivity and leukemogenesis. Having demonstrated that Class IA PI3K is hyperactivated in the presence of mutant Shp2 and contributes to GM-CSF hypersensitivity, I hypothesized the hematopoietic-specific Class IA PI3K catalytic subunit p110δ is a crucial mediator of mutant Shp2-induced PI3K hyperactivation and GM-CSF hypersensitivity in vitro and MPD development in vivo. I crossed gain-of-function mutant Shp2 D61Y inducible knockin mice, which develop fatal MPD, with mice expressing kinase-dead mutant p110δ D910A to evaluate p110δ’s role in mutant Shp2-induced GM-CSF hypersensitivity in vitro and MPD development in vivo. As a comparison, I also crossed Shp2 D61Y inducible knockin mice with mice bearing inducible knockout of the ubiquitously expressed Class IA PI3K catalytic subunit, p110α. I found that genetic interruption of p110δ, but not p110α, significantly reduced GM-CSF-stimulated hyperactivation of both the Ras-Erk and PI3K-Akt signaling pathways, and as a consequence, resulted in reduced GM-CSF-stimulated hyper-proliferation in vitro. Furthermore, I found that mice bearing genetic disruption of p110δ, but not p110α, in the presence of gain-of-function mutant Shp2 D61Y, had on average, smaller spleen sizes, suggesting that loss of p110δ activity reduced MPD severity in vivo. I also investigated the effects of three PI3K inhibitors with high specificity for p110δ, IC87114, GDC-0941, and GS-9820 (formerly known as CAL-120), on mutant Shp2-induced GM-CSF hypersensitivity. These inhibitors with high specificity for p110δ significantly reduced GM-CSF-stimulated hyperactivation of PI3K-Akt and Ras-Erk signaling and reduced GM-CSF-stimulated hyperproliferation in cells expressing gain-of-function Shp2 mutants. Collectively, these findings show that p110δ-dependent PI3K hyperactivation contributes to mutant Shp2-induced GM-CSF hypersensitivity and MPD development, and that p110δ represents a potential novel therapeutic target for JMML.

Plus de 20 million de personnes à travers le monde sont infectées par le virus T-lymphotrope humain de type 1 (HTLV-1), causant des leucémies à cellules T dans ~5 % des individus infectés. Le virus de la leucémie bovine (BLV), structurellement et fonctionnellement proche de HTLV-1, induit des leucémies à cellules B dans des modèles animaux suite à une infection naturelle (bovin) ou expérimentale (mouton). Les mécanismes moléculaires responsables du potentiel oncogène de ces deux virus restent largement incompris. Dans les deux maladies, leucémies T chez l’homme, leucémies B chez l’animal, le site intégration du virus dans les cellules leucémiques est très variable. Il est donc généralement admis que le potentiel oncogène du provirus est principalement lié à l’activité de l’oncoprotéine virale Tax. De manière paradoxale cependant, ni HTLV-1 ni BLV n’expriment de protéines virales au stade tumoral. Dans ce travail, nous avons étudié le transcriptome non codant des leucémies induites par BLV et HTLV-1 par séquençage à haut débit. Dans la première partie, nous démontrons que le provirus BLV n’est en fait pas silencieux dans les cellules tumorales. BLV produit un ensemble de dix microARNs (miRNAs) très abondants et extrêmement conservés dans toutes les tumeurs. Cette observation constitue la première description de miRNAs encodés par un rétrovirus. Les microARNs encodés par BLV sont transcrits par la RNA Polymérase III, stratégie qui permet leur production de façon indépendante de celle des messagers viraux ainsi que leur expression abondante dans le contexte tumoral caractérisé par l’absence d’activité RNA Polymérase II. Nous avons ensuite montré que, comme HTLV-1, BLV produit des transcrits encodés par le brin négatif du provirus. L’analyse par séquençage ARN à haut débit (RNA-Seq) de tumeurs induites par BLV montre l’absence d’expression virale à partir du promoteur viral situé dans le LTR 5’. Cependant, elle révèle la présence de deux transcrits viraux anti-sens non codants (AS1 et AS2) produits par le LTR 3’. Nous avons identifié ces transcrits dans toutes les tumeurs BLV analysées. Enfin, l’analyse RNA-Seq de tumeurs induites par HTLV-1 et BLV a révélé la présence d’interactions transcriptionnelles virus-hôte. Les gènes hôtes affectés sont significativement enrichis en gènes liés au cancer. Ces résultats suggèrent que les transcrits HTLV hbz et BLV AS1 jouent un rôle essentiel dans la tumorigenèse en interagissant avec le génome de l’hôte. Nous avons également détecté ce type de perturbation à des temps précoces dans le modèle expérimental BLV chez le mouton. Ces observations suggèrent que ces interactions virus-hôte constituent des événements précoces qui procurent un avantage sélectif aux clones associés, mais que d’autres altérations -génétiques et/ou épigenetiques- sont nécessaires à l’établissement de la tumeur. En conclusion, nos travaux vont permettre de mieux comprendre le rôle des interactions virus-génome hôte dans l’oncogenèse ainsi que la fonction de transcrits non codants dans le développement des cancers qu’ils soient ou non d’étiologie virale.<p><p>More than 20 million people are infected by Human T-cell Lymphotropic Virus type 1 (HTLV-1) worldwide and this will cause T-cell leukemia in 5% of them. Yet the molecular mechanisms that underlie the oncogenic potential of this virus remain largely unknown. Bovine Leukemia Virus (BLV) is closely related to HTLV1 and causes a very similar B-cell leukemia in cattle and sheep. As for HTLV1, the oncogenic mechanisms underlying BLV-induced leukemia remain poorly understood. In both diseases, leukemic cells harbor mainly one integrated provirus, yet the integration sites are very variable. As a consequence, it is generally assumed that the oncogenic effect of the provirus is largely mediated by the virally encoded Tax protein. Paradoxically, however, both HTLV1 and BLV proviruses are found to be epigenetically silenced in tumor cells. Thus Tax, as any other virally encoded protein, is not expressed in leukemic cells suggesting that other factors are involved in tumorigenesis. In this study we made three observations that might dramatically change the prevalent dogma of HTLV1 and BLV-induced leukemia. First, we demonstrated that the BLV provirus is not silent at all in tumor cells. A cluster of BLV-encoded microRNAs (miRNAs) is highly expressed, accounting for 40% of the miRNAs present in leukemic cells. This finding is the first description of retroviral-encoded miRNAs. BLV miRNAs are transcribed from five independent RNA Pol III units and are exceedingly conserved across BLV isolates (more than the protein coding genes), strongly supporting an essential yet still unknown function. Next we showed that – as HTLV1 – BLV strongly expresses antisense RNAs. High-throughput sequencing of RNA libraries (RNA-seq) from BLV associated tumors, as expected, showed no expression of viral mRNA from the 5’ LTR. However, it did reveal the presence of two novel non-coding antisense transcripts originating in the 3’ LTR of BLV. Finally, RNA-Seq analysis of HTLV-1 and BLV-induced tumors revealed that the viral 3’ LTR-driven antisense RNAs produced by both viruses interact with host genes localized in the vicinity of proviral integration. Enrichment analysis of affected host genes suggests a significant bias towards cancer-related genes. Host gene perturbations were also found at early stages post-infection in the BLV experimental model in sheep, suggesting that provirus-dependent cancer driver gene perturbations trigger initial amplification of the corresponding clones, requiring additional genetic and/or epigenetic changes to develop full blown leukemia. Overall, our findings reveal an unexpected role for BLV and HTLV antisense transcripts and contribute to the understanding of non-coding RNA-mediated mechanisms in leukemogenesis. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished